The chromosomal DNA of eukaryotic organisms is thought to be organized into a series of higher-order regions or "domains" that define discrete units of compaction of chromatin, which is the complex of nucleoproteins interacting with eukaryotic nuclear DNA. In addition to providing a means for condensing the very large chromosomes of higher eukaryotes into a small nuclear volume, the domain organization of eukaryotic chromatin may have important consequences for gene regulation. The regulation of tissue-specific gene expression at the DNA level is mediated through an interaction between regulatory sequences in the DNA of eukaryotic cells and a complex of transcriptional factors (i.e. nucleoproteins) which are specific for a particular tissue type and for a particular gene. Further, the higher-order chromatin structure of tissue-specific genes is also regulated in a tissue-specific manner (reviewed by van Holde, K. E. (1989). "Chromatin structure and transcription". In: Chromatin, K. E. van Holde, ed., New York, N.Y.; Springer-Verlag, pp. 355-408).
Higher-order chromatin domains may also define independent units of gene activity and regulation. For example, a discrete domain of eukaryotic chromatin is sometimes more than 100 kilobases in length and may encompass a particular gene or gene cluster. In those tissues where a given gene or gene cluster is active, the domain is sensitive to DNase I, thus lending support to the notion that the chromatin of an active domain is in a loose, decondensed configuration that is easily accessible to trans-acting factors (Lawson, G. M., Knoll, B. J., Marsh, C. J., Woo, S. L. C., Tsai, M-J. and O'Malley, B. W. (1982). "Definition of 5' and 3' structural boundaries of the chromatin domain containing the ovalbumin multigene family". J. Biol. Chem., 257:1501-1507; Groudine, M., Kohwi-Shigematsu, Gelinas, R., Stamatoyannoupoulos, G. and Papayannopoulou T. (1983). "Human fetal-to-adult hemoglobin switching: changes in chromatin structure of the .beta.-globin gene locus". Proc, Natl. Acad. Sci. USA, 80:7551-7555; Jantzen, K., Fritton, H. P., and Igo-Kemenes, T. (1986). "The DNase I sensitive domain of the chicken lysozyme gene spans 24kb". Nucl. Acids Res., 14:6085-6099; and Levy-Wilson, B. and Fortier, C. (1989). "The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the location of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene". J. Biol. Chem., 264: 21196-21204). By contrast, in those tissues where the same gene is not active, the chromatin of the domain is in a tight configuration that is inaccessible to transacting factors. Thus, decondensing the higher order chromatin structure of a domain is required before regulatory factors can interact with target sequences, thereby determining the transcriptional competence of that domain.
Although very little is presently known about how the higher-order chromatin structure is regulated, results from studies in physical chemistry, cell biology, and molecular biology have supported the theory that the eukaryotic genome is indeed organized into topologically isolated domains. Central to the understanding of the chromatin structure of a particular domain is how the domains are precisely defined and formed. The higher order chromatin structure of genes as well as the flanking region surrounding the genes are uniform throughout each domain, but are discontinuous in the regions, loosely termed "boundaries", between adjacent domains (Eissenberg, J. C. and Elgin, S. C. R. (1991). "Boundary function in the control of gene expression". TIG, 7:335-340). It is generally thought that domains are delimited by special nucleoprotein structures assembled at specific sites along the eukaryotic chromosome. These specific sites are believed to be the domain boundaries of chromatin.
In addition to understanding how the higher order chromatin structure of a domain is regulated as a unit, it is crucial to know how the boundaries of a domain may be organized. For example, the genome has been demonstrated to be organized into topologically isolated loops that radiate out from nuclear matrices (Benyajati, C. and Worcel, A. (1976). "Isolation, characterization and structure of the folded interphase genome of Drosophila melanogaster". Cell, 9:393-407; Paulson, J. R. and Laemmli, U.K. (1977). "The structure of histone-depleted metaphase chromosomes". Cell, 12:817-828; Gasser, S. M. and Laemmli, U.S. (1987). "A glimpse at chromosomal order". TIG;, 3:16-22; and Garrard, W. T. (1990). "Chromosomal loop organization in eukaryotic genomes". In: Nucleic Acids and Molecular Biology, F. Eckstein and D. M. J. Lilley, eds. (Berlin, Springer-Verlag) pp. 163-175). It has been suggested that the higher order chromatin structure of each of these chromatin loops is independently regulated and that the ends, or boundaries, of the loops may insulate the genes in one loop from the influence of the regulatory sequences in adjacent loops. Among the many possible functions of a boundary, the most prominent function would be that of insulating genes from the cis-acting regulatory elements of an adjacent domain.
A. Stief et al. (1989, "A Nuclear DNA Attachment Element Mediates Elevated and Position-dependent Gene Activity", Nature, 341:343-345) have reported that an "A" element, which maps to the 5' and 3' boundaries of the region of general DNase sensitivity in the active chromatin of the chicken lysozyme gene, appeared to be a type of cis-acting DNA element which possessed boundary-like properties. However, the "A" element was determined to have enhancer-like activity and to activate transcription. In addition, Stief et at. used only transient transfection assays to measure chloramphenicol acetyltransferase ("CAT") activity. Further, when the "A" element was linked to a reporter gene and transfected into chicken cells in an effort to obtain stable integration, the data presented did not portray an authentic or correlative copy number effect, since the number of the putatively integrated plasmid DNAs was measured on an absolute scale, while relative CAT activity was measured on a logarithmic scale. In fact, there was no more actual correspondence between the copy number of the reporter gene linked to the "A" element and the amount of CAT activity observed, than there was for the reporter gene not linked to the "A" element. Consequently, the chicken "A" element was neither directly nor convincingly demonstrated to be a functional or pure insulator sequence. Further, the "A" element is a strong transcriptional activator on its own and can perturb the expression of a linked gene when integrated into host DNA.
R. Kellum and P. Schedl (1992, "A Group of scs Elements Function as Domain Boundaries in an Enhancer-Blocking Assay", Mol. Cell. Biol., 12:2424-2431) described the presence of constitutively hypersensitive sites called scs (i.e. "special chromatin structures") in the fruit fly, Drosophila melanogaster. The scs, considered to be putalive boundary DNA segments of the 87A7 heat shock locus of Drosophila, were capable of blocking the action of the D. melanogaster yolk protein-1 enhancer when an scs was placed between it and the hsp70 promoter. These authors showed that the scs worked to buffer the 87A7 heat shock gene from nearby regulatory sequences in transgenic Drosophila (Kellum, R. and Schedl, P. (1991). "A position-effect assay for boundaries of higher order chromosomal domains". Cell, 64:941-950), and that the scs by itself did not possess its own regulatory activity.
However, to date, the isolation and use of a "pure" insulator from higher eukaryotes, which, on its own, does not perturb gene expression, either positively or negatively, and which serves to insulate the expression of a given gene in a mammalian system, has not been demonstrated.
In vertebrates such as chickens, mice, and humans, the beta-globin locus has been well characterized. In all three organisms, the chromatin structure of the beta-globin locus is extremely well conserved (FIG. 1 ). At the very 5' end of the beta-globin locus, a constitutive DNase I-hypersensitive site (called the 5' HS5 in humans and mice, and the 5' HS4 in chickens) is present in all tissue types (Tuan, D., Solomon, W., Li, Q. and London, I. M. (1985). "The ".beta.-like-globin" gene domain in human erythroid cells". Proc. Natl, Acad. Sci. USA, 82:6384-6388; Forrester, W. C., Takegawa, S., Papayannopoulou, T. Stamatoyannopoulos, G. and Groudine, M. (1987). "Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin expressing hybrids". Nucl. Acids Res., 15:10159-10177; and Reitman, M. and Felsenfeld, G. (1990). "Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken .beta.-globin locus". Mol. Cell. Biol., 10:2774-2786). The constitutive hypersensitive site is a DNA segment or a particular DNA sequence in a chromatin domain which is particularly sensitive to DNase I activity. Until the present invention, the function of the 5'-most constitutive hypersensitive site in the beta-globin locus of eukaryotic ehromatin was unknown.
Further into the 5' end of the beta-globin locus, there are other types of erythroid-specifie DNase I-hypersensitive sites (Tuan, D. et al., (1985), "The ".beta.-like-globin" gene domain in human erythroid cells", Proc. Natl. Acad. Sci. USA, 82:6384-6388; I. M. London et al U.S. Pat. No. 5,126,260; Grosveld, F. Blom van Assendelft, G., Greaves, D. and Killias, G. (1987). "Position-independent, high level expression of the human .beta.-globin gene in transgenie mice". Cell, 51:975-985; Forrester, W. C., Takegawa, S., Papayannopoulou, T. Stamatoyannopoulos, G. and Groudine, M. (1987). "Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin expressing hybrids". Nucl. Acids Res., 15:10159-10177; Forrester, W. C., Novak, U., Gelinas, R. and Groudine, M. (1989). "Molecular analysis of the human .beta.-globin locus activation region". Proc. Natl. Acad. Sci. USA, 86:5439-5443; Ryan, T. M., Behringer, R. R., Martin, N. C., Townes, T. M., Palmiter, R. D., and Brinster, R. L. (1989). "A single erythroid-specific DNase I super-hypersensitive site activates high levels of human .beta.-globin expression in transgenic mice". Genes & Dev. 3:314-323; and Talbot, D., Collis, P., Antoniou, M., Vida., M., Grosveld, F. and Greaves, D. R. (1989). "A dominant control region from the human .beta.-globin locus conferring integration site-independent gene expression". Nature, 338:352-355). In contrast to the 5'-most constitutive hypersensitive site, these additional hypersensitive sites may also be known as enhancer regions, or enhancers, or, as is particular to the erythroid lineage and the beta-globin locus, "locus control regions" ("LCRs") in higher eukaryotes, including mice, chickens, and humans. The beta-globin LCRs are required for a consistently high level of expression of the family of developmentally-regulated genes in the beta-globin locus. Studies using transgenic mice and DNA obtained from beta-thalassemia patients suggest that LCRs are required for decondensing the higher-order chromatin structure of the active beta-globin domain in erythroid tissues and for potently activating the expression of all of the genes in the beta-globin domain. Remarkably, the influence of the LCRs allows the decondensing of chromatin over more than 200 kilobases of DNA in the 3' direction (Elder, J. T., Forrester, W. C., Thompson, C., Mager, D., Henthorn, P., Peretz, M., Papayannopoulou, T. and Groudine, M. (1990). "Translocation of an erythroid-specific hypersensitive site in deletion-type hereditary persistence of fetal hemoglobin". Mol. Cell. Biol., 10:1382-1389); yet in both chicken and human, the chromatin upstream near the 5' constitutive hypersensitive site is believed to be in a tight, condensed configuration that is inaccessible to DNase 1.
In spite of the observations and hypotheses relating to the putative activity of insulators in nonvertebrate organisms, the isolation and functional characterization of such an element or elements in higher vertebrates, including humans, need to be achieved. Until the present invention, no authentic, pure, and functional vertebrate chromatin insulator element has been isolated or demonstrated to operate successfully as an insulator in a mammalian system. Furthermore, until the present invention, no clear, direct insulator function has been specifically ascribed to a vertebrate constitutive hypersensitive site, nor has such a pure insulator element been isolated, characterized, and functionally employed in eukaryotic and in mammalian cells.